Close Homolog of L1 (CHL1), an L1 cell adhesion molecule expressed on thalamocortical and corticofugal axons, is identified as a regulator of thalamocortical axon guidance via extracellular interactions with Neuropilin1 (Npn1) and integrins and intracellular interactions with the actin cytoskeleton. CHL1 deletion in mice disrupted the topographic projection of somatosensory thalamic axons, causing them to inappropriately target the visual cortex. This shift was first observed in the ventral telencephalon (VTe), an intermediate target with graded Sema3A expression, where axons from the ventral basal complex (VB) of the dorsal thalamus were shifted caudally. Caudal thalamic axons were also dependent upon CHL1 for preferential guidance through high Sema3A territories. A similar pattern of aberrant guidance was observed in Npn1Sema-/- mutant embryos, in which axons are nonresponsive to Sema3A. CHL1 associated with Npn1 through a sequence in the extracellular domain of CHL1, which was required for Sema3A-induced growth cone collapse. Deletion of both L1 and CHL1 further disrupted thalamocortical topography, resulting in aberrant targeting of somatosensory and motor thalamic axons to the visual cortex. For the first time, these results demonstrate a role for Sema3A, Npn1, and CHL1 in regulating the topography of thalamocortical axons in the VTe. CHL1 linkage to the cytoskeleton through ezrin, radixin, and moesin (ERM) proteins and ankyrin is critical for axon growth and guidance and cell migration. CHL1 associated with ERM proteins through a conserved sequence in the intracellular domain of CHL1, important for cell migration, neurite outgrowth and branching, and for Sema3A-mediated growth cone collapse. The cytoskeletal adaptor protein, ankyrin, was also recruited to the plasma membrane though a sequence in the CHL1 cytoplasmic domain containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Try), which was required for promoting integrin-dependent migration. CHL1 potentiated cell migration to extracellular matrix proteins by functionally interacting with [beta]1 integrins, mediated by c-Src, phosphatidylinositol 3- kinase, and ERK MAP (mitogen-activated protein) kinase. In summary, a novel role for CHL1 has been demonstrated in mediating the cellular responses of Sema3A and Npn1, important for thalamic axon guidance. In addition, the mechanism by which CHL1 associates with Npn1 and couples to the actin cytoskeleton has been defined.